The proposed study will address a novel function of Copper (Cu)-transporting ATPase (ATP7A), a Cu transporter for extracellular SOD (ecSOD), in preventing endothelial dysfunction and eNOS uncoupling in diabetes. Oxidative stress and endothelial dysfunction contribute to pathogenesis of diabetes melitus (DM), characterized by impaired insulin-Akt signaling. Although role of oxidative stress in vascular dysfunction in DM has been extensively studied, little is known about the function of antioxidant enzymes in these pathological diseases. ecSOD is a Cu-containing enzyme synthesized and secreted by vascular smooth muscle cells (VSMCs), and anchored to endothelial surfaces and extracellular matrix. Because of its extracellular location, ecSOD can increase nitric oxide (NO) bioavailability released from the endothelium by preventing O2-- mediated inactivation of NO and formation of peroxynitrite (ONOO-). Our laboratory previously demonstrated that full activation of vascular ecSOD requires Cu transporter ATP7A. However, the role of ecSOD and ATP7A in vascular dysfunction in DM is entirely unknown. Based on our preliminary studies, we will test the novel hypothesis that Akt-mediated phosphorylation of ATP7A is required for increasing ATP7A stability and translocation to the caveolae/lipid rafts where ecSOD obtains Cu to increase its activity. Thus, impaired insulin-Akt signaling in DM decreases ATP7A function and ecSOD activity in VSMCs, thereby promoting overproduction of O2- and ONOO- in the extracellular space. This may facilitate the out- side in signaling leading to eNOS uncoupling and endothelial dysfunction in ECs. Aim1 will determine the molecular mechanism by which function of ATP7A, a Cu-transporting ATPase for ecSOD, is impaired in cultured diabetic VSMC. We will test the hypothesis that insulin-Akt-mediated phosphorylation of ATP7A is required for ATP7A stabilization by inhibiting its ubiquitination/degradation as well as ATP7A translocation to caveolae/lipid rafts where ecSOD obtains Cu to promote ecSOD activity in VSMCs. Aim 2 will determine the protective role of ATP7A, a Cu-transporting ATPase for ecSOD, in eNOS uncoupling and vascular dysfunction in murine and human diabetic vessels. We will examine if vessels from DM mice or those from biopsies obtained from patients with DM show decrease in ecSOD activity and ATP7A expression, thereby increasing O2- level, ONOO- level, eNOS uncoupling, and endothelial dysfunction. We will use type1 and type2 DM mice models crossed with or without ecSOD KO, ATP7A mutant, or ATP7A overexpressing transgenic mice. We will also perform rescue experiments using adenoviral gene transfer of Akt phosphorylation sites mimetic ATP7A mutant in DM vessels ex vivo. X-ray fluorescence microscopy will be used to analyze Cu distribution in DM vessels and VSMCs. These studies should provide new insight into Cu transport system for ecSOD as a novel therapeutic strategy for treatment of oxidative stress-dependent cardiovascular diseases such as diabetes.